Acute myeloid leukemia (AMI) is a malignant disease that originates from a single transformed cell which has progressively acquired critical genetic changes that disrupt key growth-regulatory pathways. Despite the established use and optimization of regimens applying polychemotherapy and the development of multiple new agents that are effective at reducing the tumor burden in patients with leukemia, relapse continues to be the most common cause of death in AML. Newer experimental evidence demonstrates that AML arises from a small population of leukemic stem cells (LSC). Similar to normal hematopoietic stem cells (HSC), LSC are quiescent in terms of cell cycle and thus, conventional cytotoxic therapies are not effective against LSC in the majority of cases. However, therapeutic eradication of the LSC within the leukemia clone will be essential for a cure of disease. Therefore, an improved understanding of the molecular pathways that suppress the formation and maintenance of LSC is required for the development of therapies that target LSC rather than the bulk tumor cells (leukemic blasts). Recent findings demonstrate a critical role of transcription factors including PU. 1 and JUNB in the genesis and function of LSC in AML in mice and humans, and that PU.1 and JUNB are already deregulated in the early stem cell compartment. Therefore, the goals of this research project are 1) to clarify the exact mechanism of action of PU.1 and JUNB in AML-LSC, 2) to identify new target genes of PU.1 in stem cells that are important for LSC function, and 3) to identify other functionally critical alterations in the stem cell compartment that underlie the formation and maintenance of LSC in genetically defined subtypes of AML with disruption of transcription factor function. To identify implicated pathways rigorously defined stem and progenitor cell subsets will be isolated by means of multi-parameter high-speed fluorescence-activated cell sorting and then subjected to linear RNA amplification and transcriptional analysis. Identified targets will be functionally tested utilizing serial replating assays as well as murine transplantation models to assess their function in LSC. These studies will provide the basis for the development of LSC-directed therapies that might ultimately lead to a cure of AML.